L2: Enzymes involved in DNA replication and Repair

Question 1

Replication of DNA is initiated by..

Answer 1

Binding of 2 helicases (large T antigen) to origin of replication

Question 2

Helicase

Answer 2

Break HB between strands -> separate strands

Question 3

Topoisomerase

Answer 3

Remove twist/torsional stress

Question 4

Primase

Answer 4

Synthesises short RNA primers

Question 5

DNA polymerase

Answer 5

Requires primers

Copies template

Question 6

RNaseH

Answer 6

Removes RNA primers -> ozaki fragments can join

Question 7

Ligase

Answer 7

Joins nicks in lagging strand

Question 8

Synthesis of DNA starts from..

Answer 8

Small piece of RNA (primer)

Question 9

Leading strand

Answer 9

Continuous synthesis in 3’-5’ direction

Question 10

Lagging strand

Answer 10

In opp direction

Short pieces of Ozaki fragments

Question 11

DNA polymerase reaction

Answer 11

Primer strand, template strand and incoming substrate (dNTP)

dNTP -> HB with T residue in template

Question 12

Proofreading

Answer 12

DNA polymerase can move ‘back’ one nucleotide to remove incorrectly paired nucleotide

Incorrect base pairing -> DNA strand moves to exonuclease site -> incorrect base pair recognised -> nucleotide removed -> DS relocates back to polymerase site -> polymerase can incorporate correct deoxynucleoside from triphosphate substrate

3’ -> 5’ exonuclease activity of DNA polymerases I, II, III

Question 13

Active sites of DNA polymerase I

Answer 13

5’ -> 3’ polymerase site: incoming dNTP added to 3’OH group of primer -> strand grows 5’ -> 3’

3’ -> 5’ exonuclease site: strand shortens at 3’ end as enzyme moves ‘backwards’ one nucleotide

5’ -> 3’ exonuclease site: removes nucleotides in front of it as it moves forward. Can remove RNA primers

Question 14

Nick translation

Answer 14

5’ -> 3’ exonuclease activity of DNA polymerase I

Nick in DNA -> polymerase subunit can remove nucleotides &/or deoxynucleotides ahead of polymerase -> adds nucleotides as moves along

Can run along nick and remove primers

Question 15

Pol I

Answer 15

Function: ozaki fragment processing and DNA repair

Question 16

Pol II

Answer 16

Function: translation synthesis and DNA repair

Question 17

Pol III

Answer 17

Function: chromosome replication

Question 18

Pol IV & V

Answer 18

Function: Translesion synthesis

Important for replication of ‘damaged DNA’ -> stalling of replication fork. Can incorporate incorrect nucleotide opp damaged base -> replication fork proceed (translesion)

Question 19

Structure of DNA polymerase

Answer 19

Structure resembles hand with unwound 3’ end of DNA facing palm

Question 20

Incorrect base pair prevents…

Answer 20

Pol I from closing -> slow catalysis (formation of phosphodiester bond) -> enables incorrect dNTP to dissociate

Question 21

Role of metal ions in DNA polymerases

Answer 21

Active site contains 2 Mg2+ ions bound to conserved Asp residues

Helps catalysis & stabilising -ve charge from oxygen

1. Attack phosphate -> cleavage of phosophodiester linkage -> join deoxynucleotide to new strand

Deprotonates primer 3’OH -> 3’O- nucleophile

2. Stabilises pyrophosphates -> allow -ve charge on oxygen assist polymerase reaction.

Facilitates departure of pyrophosphate product

Question 22

MutS

Answer 22

Detects mismatches from distortion of backbone

Question 23

Mismatch repair by MutS

Answer 23

DNA replicated with MutS protein -> runs along looking for mutations in DNA helix from incorrect base pairing (mismatch) -> finds mismatch & recruits other proteins ( MutL & MutH) -> forms complex with MutS

MutL activated MutH -> nicks DNA strand near mutation -> exonuclease removes surrounding DNA including mismatch -> DNA repaired by Pol III & ligase

Question 24

How MutS differentiates between parent and new strand

Answer 24

Through methylation pattern on DNA

Parent strand: heavily methylated

New strand: poorly methylated as methylation takes some time after replication

Question 25

Uses of DNA in recombinant DNA technology

Answer 25

1. PCR
2. cDNA library construction
   a) ssDNA -> dsDNA
   b) mRNA copied -> ss CDNA (RNA-dependent DNA pol)
3. Nick translation.
   5’ -> 3’ exonuclease activity & DNA pol activity
4. Fill in reactions using pol activity for any DNA manipulation that cause gap in 1 strand of dsDNA